Solid phase chemistry involves chemical or biochemical reaction between components in a fluid and molecular moieties present on a substrate surface, e.g., in the synthesis of a surface-bound oligonucleotide or peptide, in the generation of combinatorial “libraries” of surface-bound molecular moieties, and in hybridization assays in which a component present in a fluid sample hybridizes to a complementary molecular moiety bound to a substrate surface. Regardless of the context, all chemical or biochemical reactions between components in a fluid and molecular moieties present on a substrate surface require that there be adequate contact between the fluid's components and the surface-bound molecular moieties. To this end, a number of approaches have been proposed to facilitate mixing of fluid components during solid phase chemical or biochemical reactions so that a substantially homogeneous fluid contacts the reactive surface. Most recently, a great deal of attention has focused on improving hybridization assays using various mixing techniques.
Hybridization reactions between surface-bound molecular probes and target molecules in a sample fluid may be used to detect the presence of particular biomaterials including biopolymers and the like. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of reacting with target molecules in solution. Such reactions form the basis for many of the methods and devices used in the new field of genomics to probe nucleic acid sequences for novel genes, gene fragments, gene variants and mutations. The ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary for elucidating metabolic pathways, for understanding biological functions, and for revealing changes in genes which confer disease. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides.
In biological chip or “biochip” arrays, a plurality of probes, at least two of which are different, are arranged in a spatially defined and physically addressable manner on a substrate surface. Such “biochip” arrays have become an increasingly important tool in the biotechnology industry and related fields, as they find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Substrate-bound biopolymer arrays, particularly oligonucleotide, DNA and RNA arrays, may be used in screening studies for determination of binding affinity and in diagnostic applications, e.g., to detect the presence of a nucleic acid containing a specific, known oligonucleotide sequence.
As array density is ever increasing, and the need for faster and more accurate hybridization assays is ongoing, there is currently a great deal of emphasis on improving “mixing” of sample fluid during hybridization and, correspondingly, in maximizing contact between the components of the sample fluid and the entirety of the array surface.
For example, the Affymetrix GeneChip® Fluidics Station hybridization and wash instrument includes a means for pumping a sample fluid back and forth across an array on a substrate surface while the substrate is mounted in a holder. While this method provides for mixing of components within the sample fluid, there are disadvantages that can adversely affect the accuracy of the hybridization reaction. That is, the method is prone to contamination because of the number and variety of materials that come into contact with the sample fluid, i.e., adhesives, various plastic components, and the like. In addition, large sample volumes (greater than 200 μl) are required, and temperature control is poor.
In U.S. Pat. No. 4,849,340 to Oberhardt, an alternative means is disclosed for mixing components in a fluid during an assay performed in an enclosed chamber. Oberhardt discloses an apparatus comprising a base, an overlay and a cover which when combined define a sample well, a channel, and a reaction space. Fluids introduced into the sample well flow by capillary action to the reaction space. Mixing of fluids within the reaction space is effected using mechanical or electromechanical means to create forced convection currents. Again, large sample volumes are required (100 to 200 μl) because of the need to maintain a gap between the base and the cover during mixing. Additionally, the method relies on capillary action to promote fluid flow, and mixing may thus be slow and incomplete, particularly when viscous reagents are used.
U.S. Pat. No. 5,192,503 to McGrath et al. discloses an apparatus for conducting an in situ assay of a tissue section mounted on a slide. A seal member, mounted on a plate, forms a closed periphery and encloses and defines an interior region on the slide that forms a reaction chamber. A plate covers the slide and seal member. The joined plate and slide together form a probe clip. The reaction chamber may comprise a single chamber or two chambers. In the one-chamber embodiment a time-release material, such as gelatin, is applied over the probe, allowing time for reaction of the tissue sample with reagents before the probe is released and thus able to react with the tissue sample. In the two-chamber embodiment, the probe reaction chamber defined by the closed periphery of a first seal member is divided into two regions by a raised portion of the plate, a mixing chamber and a reaction chamber. At least one end of this raised portion does not contact the first seal member, thereby leaving a channel available for fluid flow. Probe compounds placed in the mixing chamber do not mix with the fluid reagents in the reaction chamber until fluid is induced to flow between the two chambers via a channel in a gap left between the raised portion and the seal member. Fluid flow may be induced by rotating the probe clip to a substantially vertical orientation, allowing fluid reagents from the reaction chamber to flow into the mixing chamber and mix with the probe compounds. Re-orienting the probe clip to the horizontal causes the mixed probe and fluid reagent to flow to the reaction chamber for reaction with a tissue section therein. Thus, the position and flow of fluid reagents and probes in the reaction chamber and the mixing chamber is controlled by gravity. Optionally, both gravity-controlled flow and use of a time-release agent such as gelatin may be used at the same time to regulate the mixing of reagent fluids and probes. Like the Oberhardt device, the McGrath et al. apparatus is disadvantageous when viscous solutions are used or rapid mixing is required, insofar as mixing depends upon gravity to induce flow.
Still another method for mixing components in a sample fluid during a solid phase chemical or biochemical reaction is disclosed in commonly assigned, U.S. patent application Ser. No. 09/343,372, now U.S. Pat. No. 6,258,593 to Schembri et al., filed Jun. 30, 1999 (“Apparatus and Method The Conducting Chemical or Biochemical Reactions on a Solid Surface Within an Enclosed Chamber”). That method involves mixing a very thin film of fluid in a chamber, wherein an air bubble is incorporated therein and, when used in hybridization, a surfactant is preferably present as well.
All of the prior methods and devices of which applicants are aware are disadvantageous in one or more respects. Some of the disadvantages have been alluded to in the foregoing discussion. However, the prior art is problematic in other ways as well. For example, there has, until now, been a tradeoff between sample volume and manufacturing flexibility. That is, it is preferable to work with a very small sample volume to in order to increase the precision of the surface chemistry (as, for example, in a hybridization assay). Small sample volumes, however, have in turn meant device miniaturization, requiring extraordinarily precise control over the dimensions of all device components. Furthermore, the majority of hybridization methods and devices involve bringing the sample fluid into contact with the surface-bound probes before the correct hybridization temperature is reached; this means that hybridization will occur at non-optimum conditions, i.e., at a lower temperature, resulting in non-specific binding. In addition, contamination is frequently a problem in devices containing multiple components, and cross-contamination between samples is an additional problem with devices fabricated from non-disposable materials. Finally, sample recovery with prior devices and methods has proved difficult, as the sample fluid must be drawn off of a substrate surface after hybridization, rather than extracted from a container or well.
The present invention is addressed to the aforementioned need in the art, and provides a novel method for conducting a chemical or biochemical reaction on a solid surface as may be done, for example, in the context of a hybridization assay. The novel method provides for numerous advantages relative to the art. For example, the method:                (1) provides effective mixing during solid phase chemical or biochemical reactions and ensures that sample components adequately contact the substrate surface;        (2) allows for use of very small sample volumes without requiring correspondingly small device dimensions and associated manufacturing constraints;        (3) reduces the potential for contamination by employing disposable components and minimizing the number of different materials in contact with the sample fluid;        (4) improves the ease of sample recovery and maximizes the amount of sample fluid that can be recovered; and        (5) enables physical separation of the sample fluid and substrate surface until the desired reaction temperature (e.g., hybridization temperature) is reached.The invention thus represents a significant advance in the field of solid phase chemistry.        